Blood coagulation is controlled by a very complicated series of checks and balances such that coagulation only is triggered in the event of a bleed (Smith, Travers, & Morrissey, 2015). Injury sets off activation of these enzymes, resulting in an amplifying cascade of reactions that seals the wound. Hemophilia results from a defect in a gene coding for one of these proteins such that the cascade is aborted prematurely and bleeding continues. The most common forms of hemophilia, hemophilia A, hemophilia B and von Willebrand's disease, have long been treated by infusion of purified factor concentrates, replacing the defective enzyme and restoring the ability of the blood to clot.
Infusion of factor is remarkably effective, allowing afflicted individuals who may have died in childhood to have normal life expectancies (Hoots, 2003). With the increasing use of prophylaxis, that is, regularly scheduled infusions of factor to maintain a reasonable level of protection, these patients can lead essentially normal lives (Srivastava et al., 2012). This does not come without cost, literally and figuratively. Patients with severe hemophilia A need to infuse factor every other day due to the short circulatory half life of Factor VIII (FVIII), the protein missing in that form of the disease. This creates a number of problems, such as continued venous access and noncompliance.
Another very serious problem is encountered when patients develop inhibitory antibodies to the infused FVIII (Kempton & Meeks, 2014). About 30% of all hemophilia A patients will develop antibodies at some point in their therapy but about 5% develop such a serious inhibitor problem that FVIII infusion is no longer effective. This necessitates the use of “bypass” therapy. Factor VIIa (FVIIa) is one of the initiators of the coagulation cascade and can be used to step around the need for either FVIII or Factor IX (FIX) in the process. This requires very high concentrations of FVIIa and very frequent dosing since FVIIa has a circulatory half life of only two hours.
Because of these and other reasons, longer half-life factors are very desirable (Pipe, 2010). Less frequent dosing should improve compliance, venous access problems and expose the patient to a smaller mass of purified protein, perhaps reducing inhibitor formation. Moreover, longer half life proteins could expand treatment to the estimated 70% of hemophilia patients worldwide who are still untreated. Cost of factor is major issue but so is the complicated medical service required for hemophilia patients, particularly children. Since factor needs to be infused intravenously, rather than simply being injected subcutaneously, children with severe disease are most frequently treated at specialized hemophilia treatment centers. An obvious impediment to their treatment is that they must be delivered to the center several times per week which, in less developed countries, can put therapy beyond reach. Factors that persisted for longer periods of time could reduce these trips to once per week or even twice per month.
This problem has been recognized for some time and there have been numerous attempts to prolong the half life of factors. There are two common strategies for increasing the half life of therapeutic proteins. The first is to modify the proteins with chains of polyethylene glycol, commonly called PEGylation (Ginn, Khalili, Lever, & Brocchini, 2014). The PEG chains increase the water of hydration around the protein which results in reduced affinity for certain receptors and antibodies. The second strategy is to make use of the neonatal Fc receptor (FcRN) via fusion of the target protein with either the Fc portion of the immunoglobulins or human serum albumin (Andersen et al., 2011). Both immunoglobulins and albumin have long circulatory half lives due to their interaction with and protection by FcRN. When albumin or immunoglobulins are internalized in a variety of cells, they bind to FcRN and are recycled to the surface rather than being degraded. Both of these proteins have half lives of several weeks as a result.
These strategies have been successfully utilized to increase the half life of human Factor IX, the protein involved in hemophilia B (Mannucci & Mancuso, 2014). They have been less successful in prolonging the half life of FVIII (Buyue et al., 2014; Stennicke et al., 2013). FVIII itself is an unstable protein and requires the presence of von Willebrand Factor (vWF). FVIII in the absence of vWF has a half life of only a few minutes. The half life of the complex is determined by the half life of vWF so modifications to FVIII have only a small effect, increasing half-life from 12 hours to 18 hours.
Similar strategies have been attempted for FVIIa including PEGylation, fusion to albumin and Fc (Oldenburg & Albert, 2014; Schulte, 2008; van der Flier et al., 2015). Each of these modifications increased the half-life from 2 hours to over 10 hours but failed to solve another issue with FVIIa. FVIIa is most active in complex with Tissue Factor (TF). In the absence of TF, very high concentrations of FVIIa are needed to effect hemostasis. In some cases of engineered FVIIa, this has resulted in inhibitor formation.
Accordingly, there is a need for compositions and methods for long half-life coagulation complexes.